Assuntos
Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Estabilidade Enzimática , Magnésio/metabolismo , Manganês/metabolismo , Vírus da Leucemia Murina de Moloney/enzimologia , Vírus da Leucemia Murina de Moloney/genética , Engenharia de Proteínas , RNA Helicases/genética , RNA Helicases/metabolismo , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/estatística & dados numéricos , Sensibilidade e Especificidade , Thermococcus/enzimologia , Thermococcus/genéticaRESUMO
Detection of mRNA is a valuable method for monitoring the specific gene expression. In this study, we devised a novel cDNA synthesis method using three enzymes, the genetically engineered thermostable variant of reverse transcriptase (RT), MM4 (E286R/E302K/L435R/D524A) from Moloney murine leukemia virus (MMLV), the genetically engineered variant of family A DNA polymerase with RT activity, K4polL329A from thermophilic Thermotoga petrophila K4, and the DNA/RNA helicase Tk-EshA from a hyperthermophilic archaeon Thermococcus kodakarensis. By optimizing assay conditions for three enzymes using Taguchi's method, 100 to 1000-fold higher sensitivity was achieved for cDNA synthesis than conventional assay condition using only RT. Our results suggest that DNA polymerase with RT activity and DNA/RNA helicase are useful to increase the sensitivity of cDNA synthesis.
Assuntos
DNA Complementar/biossíntese , DNA Complementar/genética , RNA/análise , RNA/genética , Sequência de Bases , DNA Helicases/genética , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Estabilidade Enzimática , Expressão Gênica , Bacilos Gram-Negativos Anaeróbios Retos, Helicoidais e Curvos/enzimologia , Bacilos Gram-Negativos Anaeróbios Retos, Helicoidais e Curvos/genética , Vírus da Leucemia Murina de Moloney/enzimologia , Vírus da Leucemia Murina de Moloney/genética , Análise de Sequência com Séries de Oligonucleotídeos , Engenharia de Proteínas , RNA Helicases/genética , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura , Thermococcus/enzimologia , Thermococcus/genéticaRESUMO
A switch of viral hemagglutinin receptor binding specificity from bird-type α2,3- to human-type α2,6-linked sialic acid is necessary for an avian influenza virus to become a pandemic virus. In this study, an easy-to-use strip test to detect receptor binding specificity of influenza virus was developed. A biotinylated anti-hemagglutinin antibody that bound a broad range of group 1 influenza A viruses and latex-conjugated α2,3 (blue) and α2,6 (red) sialylglycopolymers were used in an immunochromatographic strip test, with avidin and lectin immobilized on a nitrocellulose membrane at test and control lines, respectively. Accumulation of a sialylglycopolymer-virus-antibody complex at the test line was visualized by eye. The strip test could be completed in 30min and did not require special equipment or skills, thereby avoiding some disadvantages of current methods for analyzing receptor binding specificity of influenza virus. The strip test could detect the receptor binding specificity of a wide range of influenza viruses, as well as small increases in the binding affinity of variant H5N1 viruses to α2,6 sialylglycans at viral titers >128 hemagglutination units. The strip test results were in agreement with those of ELISA virus binding assays, with correlations >0.95. In conclusion, the immunochromatographic strip test developed in this study should be useful for monitoring potential changes in the receptor binding specificity of group 1 influenza A viruses in the field.
Assuntos
Aves/virologia , Cromatografia de Afinidade/instrumentação , Vírus da Influenza A/isolamento & purificação , Influenza Aviária/diagnóstico , Fitas Reagentes/análise , Animais , Cromatografia de Afinidade/economia , Desenho de Equipamento , Glicoproteínas de Hemaglutininação de Vírus da Influenza/isolamento & purificação , Humanos , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Humana/diagnósticoRESUMO
BACKGROUND: Recombinant adeno-associated virus vectors based on serotype 2 (AAV-2) have become leading vehicles for gene therapy. Most humans in the general population have anti-AAV-2 antibodies as a result of naturally acquired infections. Pre-existing immunity to AAV-2 might affect the functional and safety consequences of AAV-2 vector-mediated gene transfer in clinical applications. METHODS: An enzyme-linked immunosorbent assay (ELISA) method was developed using microwell plates coated with intact particles of recombinant AAV-2 vectors, and horseradish peroxidase-conjugated anti-human immunoglobulin G (HRP-IgG). Neutralizing antibody titres were analysed by assessing the ability of serum antibody to inhibit transduction into HEK293 cells of AAV vectors that express beta-galactosidase. RESULTS: Anti-AAV-2 antibodies were detected by ELISA in two of 20 healthy subjects. The positivity criterion (optical density >0.5) in ELISA corresponded to the cut-off value (320-fold dilution of serum) in the AAV-2 neutralization assay. Influences of interfering substances were not observed. CONCLUSION: This ELISA method may be useful for rapid screening of anti-AAV-2 neutralizing antibodies in candidates for gene therapy.
Assuntos
Anticorpos Neutralizantes/química , Química Clínica/métodos , Dependovirus/genética , Ensaio de Imunoadsorção Enzimática/métodos , Terapia Genética/métodos , Linhagem Celular , Técnicas de Transferência de Genes , Vetores Genéticos , Peroxidase do Rábano Silvestre/genética , Humanos , Imunoglobulina G/genética , Testes de Neutralização , Valores de Referência , beta-Galactosidase/metabolismoRESUMO
The SSX family proteins have been considered new members of the cancer/testis antigens because of the restricted expression in testis among normal tissues and the activation in a wide range of cancers. Thus, they would be potential molecular targets for immunotherapeutic strategies. We have developed a competitive nucleic acid sequence-based amplification (NASBA) assay to analyze SSX mRNA expression in 211 bone and soft tissue tumors. The copy numbers of SSX mRNA per mug of total RNA in tumor tissues were widely distributed, ranging logarithmically from 0.6 to 6.6. We found that malignant tumors showed significantly higher expression of SSX mRNA than benign tumors (P < 0.0001). Further, SSX mRNA expression in stage III tumors was significantly higher than that in stage I or II tumors (P < 0.005). This NASBA assay was also more sensitive compared to immunohistochemistry using newly affinity-purified polyclonal antibody against SSX. Collectively, these results suggest that the SSX quantitative NASBA assay could provide useful information to select eligible patients for SSX-specific cancer vaccines.
Assuntos
Neoplasias Ósseas/genética , Proteínas de Neoplasias/genética , RNA Mensageiro/análise , Proteínas Repressoras/genética , Replicação de Sequência Autossustentável , Neoplasias de Tecidos Moles/genética , Adolescente , Adulto , Idoso , Animais , Neoplasias Ósseas/imunologia , Células COS , Criança , Feminino , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/análise , RNA Mensageiro/metabolismo , RNA Neoplásico/análise , RNA Neoplásico/metabolismo , Proteínas Repressoras/análise , Neoplasias de Tecidos Moles/imunologiaRESUMO
Accurate quantification of multidrug resistance-1 gene (MDR1) expression in target cells would be of important therapeutic value in predicting cellular response to anticancer drugs. Because certain normal cells in peripheral blood physiologically express MDR1, increasing the sensitivity of the detection methods might result in confounding low-degree expression in tumor cells with physiologic expression in normal cells. The purpose of this study was to determine MDR1 mRNA expression levels in peripheral blood leukocytes obtained from healthy adult volunteers using a competitive nucleic acid sequence-based amplification (NASBA) assay. We determined the reference intervals of MDR1 mRNA expression in peripheral blood obtained from 98 healthy adults by measuring its expression with the quantitative NASBA assay between 5.50 x 10(4) copies/microg RNA and 6.76 x 10(5) copies/microg RNA. The new reference intervals were evaluated using a number of sensitive or resistant cell lines as control; positive or negative MDR1 expression was clearly demonstrated. We also reevaluated MDR1 expression levels in leukemia cells obtained from patient peripheral blood; 18 of 31 samples (58%) exceeded the newly established upper reference limit. The cutoff value established could be used to distinguish significant MDR1 expression in tumor cells from physiologic expression in certain normal cells coexistent in peripheral blood.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Leucócitos Mononucleares/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Adulto , Estudos de Casos e Controles , Expressão Gênica , Humanos , RNA Mensageiro/análise , Replicação de Sequência Autossustentável/métodos , Células Tumorais CultivadasRESUMO
BACKGROUND: Because clinical drug resistance is caused by low-grade expression of a responsible gene, highly sensitive methods are desirable for its detection in clinical settings. We developed a quantitative nucleic acid sequence-based amplification (NASBA) assay for multidrug resistance-1 (MDR1) transcripts, and applied it to clinical samples. METHOD: MDR1 transcripts were amplified using the NASBA technique combined with sandwich hybridization of amplified MDR1 mRNA followed by chemiluminescence detection on an automated analyzer. Quantification of MDR1 mRNA was achieved through competitive coamplification of in vitro-generated RNA, which acts as an internal control. RESULTS: The competitive NASBA assay exhibited higher sensitivity (reliable detection limit was 100 copies of MDR1 mRNA) and linearity over a broader dynamic range (7 logarithmic orders) than the competitive reverse transcription-polymerase chain reaction assay. All 33 clinical samples obtained from patients with leukemia were successfully assayed, demonstrating its feasibility. MDR1 expression-compensated with beta-actin expression-ranged from 1.4 x 10(2) to 2.5 x 10(6) (median 4.8 x 10(5)) copies/microg RNA, while the range of MDR1 expression in peripheral blood samples from 15 healthy adults was from 8.9 x 10(4) to 5.2 x 10(5) (median 2.2 x 10(5)) copies/microg RNA. MDR1 expression in 8 of 33 clinical samples exceeded the median of healthy adult samples. CONCLUSIONS: The competitive NASBA assay is applicable to MDR1 mRNA quantification in clinical samples and would contribute to detection of clinical multidrug resistance.
